1. Field of the Invention
The molecules and methods of the present invention provide a means for expressing a heterologous gene in a selected subset of cells. The precursor-therapeutic molecules (PTM) of the invention are substrates for a targeted or defined trans-splicing reaction between the precursor therapeutic molecules and pre-mRNA molecules which are uniquely expressed in the specific target cells. The PTMs may be RNA, DNA, or other molecules such as peptide nucleic acids (PNA). The in vivo trans-splicing reaction provides an active therapeutic molecule which may be expressed in the target cells. The expression product of the mRNA may be a protein of therapeutic value to the cell, or a toxin which kills the specific cells. Alternatively, the therapeutic RNA (th-RNA) or other molecule may itself perform a therapeutic function. On another embodiment of the invention multiple PTMs may be used in combination to achieve a therapeutic effect.
2. Description of Related Art
One of the greatest challenges in the therapy of many life-threatening disease conditions, such as cancer and AIDS, is the administration of a therapeutic molecule to the specific target cells without administering the same molecule to other cells in the organism. Previous efforts to solve this problem have included gene therapy with viral vectors, delivery of drugs or toxins conjugated to monoclonal antibodies, and others. To date, these methods have not been entirely effective.
The method of the present invention does not require delivery to only the targeted cells. The precursor molecule to the therapeutic molecule can be delivered to all cells in the organism, and may be taken up by all cells in the organism, but the therapeutic mRNA is only created in vivo in the specific target cells. The specificity of this therapy relies on the unique (restricted) transcription of the target pre-mRNA in the target cells. The normal cells (non-targeted) will not transcribe (or transcribe only minimally) the target gene. Therefore, selective creation of the therapeutic molecule will not take place in such normal cells (or will only take place to a very minimal extent).
One important way that eucaryotic cells in the same organism differ from one another, despite virtual identity of gene content, is that they express different genes or portions of those genes. This regulation of gene expression operates at many levels; classical studies on gene expression demonstrate control at the level of transcription and translation. More recent work indicates that cells also have the ability to regulate gene expression by gene copy number and regulation of splicing.
The genes, stored as DNA sequences in the chromosome, are transcribed into pre-mRNAs which contain coding regions (exons) and generally also contain intervening non-coding regions (introns). The pre-mRNA is processed in the nucleus, removing the introns, along with any unwanted exons. The remaining exons are spliced together, forming an mRNA, which is exported from the nucleus to the cytoplasm for translation into a protein by the ribosomes. See, for example, Moore, M. J., C. C. Query, and P. A. Sharp, Cell, 77:805-815 (1994); Moore, M. J., C. C. Query, and P. A. Sharp, The RNA World, Cold Spring Harbor Laboratory Press, 303-358, (1993).
Introns are removed from pre-mRNAs in a precise process called splicing. Chow, L. T., R. E. Gelinas, T. R. Broker, R. J. Roberts, (1977) Cell, 12, 1-8; and Berget, S. M., C. Moore and P. A. Sharp (1977) Proc. Natl. Acad. Sci. USA 74, 3171-3175. Pre-mRNA splicing proceeds by a two-step mechanism. In the first step, the 5' splice site is cleaved, resulting in a "free" 5' exon and a lariat intermediate. (Moore, M. J. and P. A. Sharp, Nature, 365:364-368, 1993) The 5' nucleotide of the intron (usually guanine) forms the lariat intermediate through a 2',5'-phosphodiester link with the branch point nucleotide (usually adenosine) in the intron. In the second step, the 5' exon is ligated to the 3' exon with release of the intron as the lariat product. These steps are catalyzed in a complex of small nuclear ribonucleoproteins and proteins called the spliceosome (Moore et al., The RNA World).
The trans-esterification splicing reaction sites are defined by consensus sequences around the 5' and 3' splice sites. The 5' splice site consensus sequence is AG/GURAGU (where N=any nucleotide, A=adenosine, U=uracil, G=guanine, C=cytosine, R=purine, Y=pyrimidine, and /=the splice site). Moore et al., The RNA World. The underlined nucleotides are common to almost all pre-mRNA introns, with GC substituted in place of GU being a rare exception. The 3' splice site consists of three separate sequence elements: the branch point or branch site, a polypyrimidine tract and the 3' consensus sequence. These elements loosely define a 3' splice site region, which may encompass 100 nucleotides of the intron upstream of the 3' splice site. The branch point consensus sequence in mammals is YNCUGAC. The underlined A is the site of branch formation (the BPA=branch point adenosine). The 3' splice consensus sequence is YAG/G. Between the branch point and the splice site there is usually found a polypyrimidine tract, which is important in mammalian systems for efficient branch point utilization and 3' splice site recognition (Roscigno, R., M. Weiner and M. A. Garcia-Blanco, J. Biol. Chem. 268, 14, 11222-11229, 1993). The first YAG dinucleotide downstream from the branch point and polypyrimidine tract is the most commonly used 3' splice site. Smith, C. W. J., E. B. Porro, J. G. Patton and B. Nadal-Ginand (1989) Nature 342, 243-247.
Cis vs. trans-splicing
Usually exons are ligated to other exons in the same pre-mRNA, cis-splicing, and not to exons in other pre-mRNAs, trans-splicing. It is possible, however, to observe efficient trans-splicing in vitro by tethering two halves of a pre-mRNA using complementary sequences. These form stable double stranded stems by "Watson-Crick"-like base pairing. Konarska, M. M., R. A. Padgett and P. A. Sharp (1985), Cell 42, 165-171. This type of trans-splicing has not been clearly observed in vivo.
The mechanism of splice site approximation "through space" is independent of the intron between the splice sites for example in the trans-splicing observed commonly in trypanosomes and nematodes. Sutton, R. E. and J. C. Boothroyd, Cell 47, 527-535 (1986); Murphy, W. J. et al., Cell 47, 517-525 (1986); Krause, M. and D. Hirsh, Cell 49, 753-761 (1987). In these very special cases, the 5' splice site containing short leader (SL) RNA forms a small nuclear ribonucleoprotein particle (snRNP) that interacts with the 3' end of the intron in the larger pre-mRNA (Bruzik, J. P. and J. A. Steitz, Cell 62, 889-899 (1990); Bruzik, J. P. and T. Maniatis, Nature 360, 692-695 (1992). This type of splicing has not been observed to occur naturally in mammalian cells.
Konarska et al. (1985), D. Solnik (1985) Cell 42, 157-164 detected trans-splicing in vitro using RNAs that did not resemble the SL RNAs. RNA-RNA secondary structures which tethered the precursors significantly increased the efficiency of the trans-splicing reaction (from &lt;1% to 15-30% of wild type cis-splicing efficiency).
Complementary RNA or DNA sequences can specifically base pair with unique target sequences of RNA or DNA. The specificity of binding is influenced by the sequence, the length of the complementary region, and any secondary structure at the binding site. In order to obtain binding specificity, a unique sequence is chosen as the target. A chain length of 17 nucleotides has been calculated to be sufficient to achieve binding specificity, that is, the statistical single occurrence of a unique polynucleotide target in the human haploid genome of 3.times.10.sup.9 base pairs. M. Smith, Methods of DNA and RNA Sequencing, ed S. M. Weissman, Praeger, New York, N.Y., USA, p. 39 (1983). Duplex stability is independent of length for complementary sequences longer than 200 nucleotides [Steiner, R. F. and Beers, R. J. Jr. (1986)]. In Polynucleotides, (Elsevier, Amsterdam). Longer complementary sequences increase the stability of the duplex, but very long regions can interact with multiple mRNAs through base pairing involving only 510 contiguous bases, thus lowering their specificity. Complementary sequences may have non-binding RNA or DNA sequences or other nucleic acid analogs or chemical groups on either of their 5' and/or 3' ends. Binding may also be achieved through other mechanisms, for example triple helix formation, and protein-nucleic acid interactions, such as those between gene promoters and DNA. Examples of tissue specific promoters include the immunoglobulin promoter described by Brinster at. al., Nature, 306:332-336 (1983) and the insulin promoter described by Bucchini et. al., PNAS, 83:2511-2515 (1986). Other means of binding may be used which are known to those skilled in the art.
Toxins such as diphtheria toxin (DT), ricin, Pseudomonas toxin, shiga toxin, and cholera toxin are extremely potent. A single molecule of DT can kill a cell by acting enzymatically within the cytosol. Yamaizumi, M., E. Mekada, T. Uchida and Y. Okada, Cell 15, 245-250 (1978). These toxins appear to have a similar basic structure, consisting of an A and B subunit, wherein the B subunit binds to the cell surface and facilitates the translocation of the A subunit into the cell, and the A subunit possesses the enzymatic toxin activity. Collier, R. and J. Kondel, Biol. Chem. 246, 1496-1503 (1971); and Gill, D. and A. Pappenheimer, J. Biol. Chem. 246, 1492-1495 (1971). The DT A subunit (DT-A) catalyzes the transfer of ADP-ribose from NAD to an unusual amino acid (dipthamide) in elongation factor 2. Honjo, T., Y. Nishizuka and 0. Hayaishi, J. Biol. Chem. 243, 3553-3555 (1968); and Gill, D., A. Pappenheimer, R. Brown and J. Kurnick, J. of Experimental Medicine 129, 1-21 (1969). Such binding stops protein synthesis in the cell and is lethal to that cell. There are a number of therapeutic strategies which attempt to deliver or express the DT-A within selected cells, including transcriptionally regulating DT-A gene expression. (Robinson, D. F., T. H. Maxwell, Hum. Gene Ther., 6(2), 137-145, 1995; Cook D. R. et al., Cancer Biother., 9(2), 131-141, 1994; Curiel, T. J. et al., Hum. Gene Ther., 4(6), 741-747, 1993).